Preparation of spiramycin iii



Twas.

United States Lbn Ninet, Paris, and Jean V'errier, Boulogne-sur-Seine,

France, assignors to Societe des Usines Chimiques Rhones-Poulenc, Paris,France, a corporation of France No Drawing. Filed Apr. 11, 1958, Ser.No. 727,765 Claims priority, application France Apr. 19, 1957 16 Claims.Cl. 195-80) This invention relates to an improved process for thepreparation of the antibiotics known as spiramycins II and III, thetherapeutic activity of which, in human therapy is verified in the fileof application Serial No. 558,753, filed January 12, 1956.

Spiramycin, an antibiotic composed of three constituents of very similarproperties respectively known as spiramycin I, II and III is produced bythe culture of a strain of Streptomyces am bofaciens, a specimen ofWhich has been deposited at the NRRL, Peoria, Illinois, United States ofAmerica, where it has been given the designation NRRL 2420, or one ofits mutants, in the appropriate culture medium. The organism isidentified as Streptomyces am'bofaciens NRRL 2420.

The exact constitution of these spiramycins is'still not known withcertainty but it has been found that spiramycins II and III arerespectively the acetyl and propionyl derivatives of spiramycin I.Spiramycins II and III can. be crystallised, which facilitates theirpurification and constitutes an important advantage in theirpharmaceutical use.

An important advantage of spiramycins II and III is their lower toxicityas compared with spiramycin I. This is shown in the following table ofthe doses (expressed in g./kg.) of the three spiramycins which produce a50% mortality rate in mice treated by the subcutaneous route (LD g./kg.s.c.).

Products LD (g /kg. so.)

Splramyein L 1. 01' Spiramyciu IL 1. 52 spiramycin IIL 2. 04

it is possible to atiect the proportion of the three spiramycins presentin the product produced by Streptomyces ambofaciens NRRL 2420.Nevertheless with the usual media used for the preparation ofspiramycins, which are media based on ill-defined and complex substancesor purely synthetic media, the spiramycin I content of the spiramycinproduced remains rather high. As a result it is very diificult to obtainspiramycins II and III free from spiramycin I otherwise than in ratherpoor yields.

It has been discovered, and this forms the basis of the presentinvention, that by operating under suitable conditions it is possible todirect the fermentation of Streptomyces ambofaciens NRRL 2420 or itsmutants in such a way that there is obtained a considerable increase inthe relative amounts of spiramycin II and/ or spiramycin III in thetotal spiramycin produced by the microorganism. In the case ofspiramycin III, for example, a proportion of to by weight of the totalspiramycin may be obtained as spiramycin HI. It has also been found thatStreptomyces ambofaciens NRRL 2420 or its quality of the acylatingenzymatic system can be varied Patented June, 28;, 19260,

mutants are capable of converting spiramycin I into spiramycins II andIII by an enzymatic acetylation or propionylation; inthis way thetransformation ofspiramycin I can be completedunder the best conditions.

According tov the present inventiona process for the production ofspiramycin H or spiramycin III or mixtures thereof comprises culturingStreptomyces ambojaciens NRRL 2420 under aerobic conditions on anutrient culture medium in the. presence of an acetylating orpropionylating agent or precursor therefor added to the medium. The.culture may be. carriedout on nutrient media known per se or-on nutrientmedia already containing spiramycin L t V Asacetylat-ing agents theremaybe used acetic acid, its salts, e.g. its alkali metal salts, itsesters or acetamide. There may also be used compounds which under theconditions of the culture generate an acetylating agent tor examplebutyric. acid and its-derivatives. Such a compound is hereinafterreferred to as a precursor. For reasons of convenience. it is preferredto employ acetic acid or sodium acetate at a concentration of between0.1 and 30 g. per litre in the medium.

As propionylating agent there may be used 'propionic of 2-15 g. perlitre, The acylating agent can be added as an aqueous solution or asolution in an organic solvent such, as methanol, ethanol, acetone,benzene, diethyl ether or dichlorethane. The acylating agent may ifdesired be added in. successive small quantities without producing anyimportant modifications in the desired transformatiom In the usualcomplex or synthetic media the fermentation does not present anydifiiculties and proceeds normally. i

Where a medium containing spiramycin I is employed it is desirable totake account of the dilferent factors which favour biochemicaltransformationof spiramycin I into spiramycins II and III. It isnecessary for best results to cultivate Streptomyces ambofaciens' NRRL:

2420 in conditions which permit at the same time development and thecultivation of the enzymatic system. Further, it is convenient toutilise the transforming capacity acquired by the culture by employingit as an inoculum medium for addition to media containing spiramycin I,

Thus Streptomyces ambofaciens NRRL 2420 can be cultivated in a culturemedium containing initially the spiramycin I to be transformed as Wellas all the necessary factors for the production of the enzymatic systemand for the transformation, by the system, of the spiramycin I presentinto spiramycins II and III. It has however been found particularlyadvantageous in carrying out the present. invention first to cultivateStreptomyc'es ambofaeiens NRRL 2420 under conditions such that itproduces the acylating enzymatic system and then, as a second step, toutilise the transforming. capacity acquired by the organism by puttingits mycelium intov contact with spiramycinl and the necessary elementsfor the transformation, i.e. substances capable. of producing acetyl orpropionyl radicals, and activators.

The culture of Streptomyces ambofaciens NRRL 2420 with a. view to theproduction of an acylating enzymatic system can be carried out on verydiverse media, in par ticular on the media which are usually used i.e.complex media or synthetic media; However the quantity and 3 over a widerange according to the compositions of the media used. Also, in order toobtain a mycelium possessing a high capacity for transforming spiramycinI into. spiramycinsll and III, it is preferable to select very carefullythe various products entering into the composttion of the medium.

It has been found particularly advantageous to use a synthetic mediumcontaining only a glucidic source of energy, a source of ammoniacalnitrogen and neutralising substances. amounts varying from 1 to 100g./1., the optimum being between 40 and 60 g./l. Among the ammoniumsalts, the chloride, sulphate or nitrate are preferred. The quantity ofammoniacal nitrogen can be varied between 0.1 and g./l., the optimumbeing between 2 and 4 g./l.

The initial pH of the culture of Streptomyces ambofaciens NRRL 2420should be adjusted to between 5 and 9 and preferablybetween 6.5 and 7.5in order to achieve a product of high activity in the transformation ofspiramycin I.

To avoid change of pH of the culture medium to values which are too lowand which may inhibit a high yield of the enzymatic acylating system itis convenient to add to the culture medium neutralising substances inadequate quantity to maintain the pH at the desired value. For thispurpose there may be used alkaline agents such as alkali and alkalineearth metal hydroxides and carbonates in solution or suspension inwater. It is also possible to add, before the commencement of theculture operation, a potential neutralising substance such as aninsoluble carbonate of an alkaline earth metal. Preferably calciumcarbonate is used in amounts between 1 and 50 g./l., the optimum amountbeing about 25 g./l.

It is similarly preferred to cultivate Streptomyces ambafaciens NRRL2420 under well-defined temperature conditions in order to obtain themaximum production of the acylating enzymatic system. The culture canfor example be carried out between and 40 C. and preferably be tween23-27 C. The culture of Streptomyces ambafaciens NRRL 2420 can becarried out under the usual conditions required for aerobic fermentationand more especially in apparatus which allows both a good dispersion ofthe necessary for the respiration of streptomyces and a goodhomogenisation of the culture.

It has been found that the transforming capacity of the Streptomycesambofaciens NRRL 2420 cultures varies.

with the age of the culture. At the beginning of culturing thetransforming capacity is practically proportional to the quantity ofmycelium present in the cultures. After a certain time thetransformation capacity attains a maximum value and then decreases,while the mycelium continues its growth. In practice it has been foundthat the mycelium possesses the maximum transformation capacity after30-150 hours of culture, more often between 40 and 100 hours, theoptimum time depending essentially upon the general conditions of theculture.

After having prepared, as will be described, a culture of Streptcmycesambofaciens NRRL 2420 or one of its mutants, in order to produce theenzymatic acylating system which converts spiramycin I into spiramycinsII and III it is then necessary to place the culture in conditions whichwill utilise its transforming capacity. There can be added to theculture obtained both the spiramycin I to be transformed and thenecessary elements for the transformation, i.e. the acylating agents andactivators. The mycelium of Streptomyces ambofaciens NRRL 2420 can alsobe isolated and placed in a suitable transforming medium containingspiramycin I, the acylating agents and activators.

It has been found that the proportion of transformation products ofspiramycin I obtained varies according to the nature of the acylradicals present in the transforming medium. Thus propionyl radicals areattached more easily to the spiramycin 1 than acetyl radicals.

Preferably glucose or starch is used in Each time that the two types ofradicals are simultaneously present in the transformation medium there1s a preferential utilisation of the propionyl radicals to convertspiramycin I to spiramycin III. Thus the transformation of spiramycin Iintospiramycin III can be facilitated by the use of substances capableof supplying an excess of propionyl radical. The specific transformationof spiramycin I to spiramycin II only occurs in the absence from thetransforming medium of all substances capable of supplying the propionylradical. Since Streptomyces ambofaciens NRRL 2420 produces, with verygreat ease and in the widest range of media, substances capable ofsupplying a propionyl radical, the specific transformation of spiramycinI exclusively to spiramycin II can only be carried out under controlledconditions.

If the cultures produced in order to support the acylating system areused directly as transformation media, practically pure spiramycin IIIis then obtained by the addition of a propionylating agent. But by theadditionof an acetylating agent spiramycin I is only converted into amixture of spiramycins II and III because of the pres-- ence in themedium of small quantities of propionylating agent.

, However, if the mycelium of the Strepfomyces ambafaciens NRRL 2420culture is separated from the medium which has been used to produce theenzymatic acylating system, it is then possible to effect a close con-'trol over the transformation of spiramycin I. It is suflicient in effectto add to the mycelium, in addition to the optional activators, thesubstances capable of supplying acyl radicals. According to the natureof the latter, spiramycin II, spiramycin III or a mixture of the two isthen obtained; This process is particularly advantageous for theproduction of spiramycin II, which can only be obtained in the presenceof substances capable of suppl ing acetyl radicals to the exclusion ofsubstances capable of supplying propionyl radicals, particularly thosewhich arise from metabolism of Streptomyoes ambofaciens NRRL 2420.

spiramycin I can be used in the pure state or in a mixture withspiramycins II and III. Such a mixture is obtained by the usualprocesses of production of crude spiramycin. The spiramycin can be usedin the form of the base or a salt and added to the transformation mediumin solid or dissolved state. tion spiramycin can be used, for example,in the form of the base dissolved in water, methanol, ethanol, diethylether, acetone, benzene or dichlorethane. It can also be used as a salt,more especially the hydrochloride, sulphate, acetate or propionate inaqueous solution.

It is preferable that the concentration of the solutions of spiramycinadded to the transforming medium should be such that there is nosubstantial dilution of the latter medium. The final concentration ofthe spiramycin I in the transforming medium may be varied, for example,between 1 and 50 g./l. but it is particularly advantageous to use aconcentration of spiramycin I between 5 and 20 g./l. V Thisconcentration can be obtained by a single additron of spiramycin I or bythe addition of several fractions without producing important variationsin the desired transformation product.

As a result of the preceding considerations it will be appreciated thata culture of Streptomyces ambofaciens NRRL 2420 possessing an enzymaticacylating system with the optional addition of acylating agents, can byitself produce the enzymatic transformation of spiramycin I which is theobject of the present invention.

Theaddition of mineral or organic salts does not usually lead to anyimportant variation in the quantity of spiramycin I transformed.Nevertheless it has been found that certain anions and cations have afavourableetfect on the transformation of spiramycin I into spiramycinsII and III, either by accelerating the transformation or by increasingthe quantity of spiramycin converted.

For ease of manipula-- Among the anions possessing activating propertiesin the enzymatic reaction is chlorine, which may be added in Amongcations which are.capable,..of activating the transformation aremagnesium, ironand cobalt, thislast being the activator of choice forthe enzymatic. acylating, reaction. These metals can be-addedinthe-formof-chlmride, sulphate, nitrate or any-other..salts.-;-whichrdo not hinder the enzymatic reaction. "Elms,for-,exatnplm;rnag

nesium and iron can be used in amounts between 0.1 and 500 mg. per litrein the transforming medium, the optimum being between 5 and mg. /l.Cobaltcanbeused, for example, in amounts between 0.11 and1l00Omg./'l.=the optimum being between 10 and 400'mg./'li

In order to effect the transformation ofispiramycinl into spiramycins IIand III under the best possible conch-F tions it, is therefore necessaryto. add-to the culture of Streptomyces ambofaciens NRRL 2420 possessingthe enzymatic acylating system, the spiramycin I to be transformed,acylating agents and activators as aforesaid.

These different elements can be added simultaneously or separated intime. It is nevertheless preferable toadd the spiramycin I after theacylating agents and activators. It has been found advantageous incertain cases to add, atthe same time as spiramycin I the acylatingagents and activators, a source of energy such as glucose or starch.This is of importance especially where the mycelium of Streptomycesambofaciens NRRL 2420 is separated from its culture medium and is placedin a medium containing only spiramycin, acylating agents and activators.Thus, for example, glucose or starch may be added in a quantity between1 and 50 g./l., the optimum being about 10 g./l.

When the culture containing the enzymatic acylating system is itselfused as the transforming medium the addition of the different elementsis preferably effected at a pH between 5 and 8 and most advantageouslybetween 6 and 7. It is sometimes desirable, in order to carry out thetransformation under the best possible conditions, to modify the pH ofthe culture at the moment of addition of the various elements. To thisend acid or alkaline substances can be used which have substantially noinhibiting properties on the transformation reaction. For example,-hydrocbl-oric, sulphuric, nitric, acetic, propionic or citric acids orsodium, potassium, ammonium, calcium or barium hydroxides may be used.

When. the transformation is carried out by separating the \mycelium ofSlreplomyces ambofaciens NRRL 2420 from the medium and placing themycelium in the presence of spiramycin, acylating agents, activators andsources of energy, it is advantageous to regulate the pH between 3 and 8with the aid of acidic or basic agents as justv described. The pH canalso be regulated with the aid of buffering media such as aceticacid-acetate, proponic acid-propionate, citric acid-citrate and phthalicacidphthalate mixtures.

In addition, whatever mode of transformation is selected, it may beadvantageous to correct the variations in pH which are produced in thecourse of the transformation by addition of acidic or basic agents orbuffering mixtures.

The transformation of spiramycin I into spiramycins II and III byStreptomyces ambofaciens NRRL 2420 continues while the culture of thisorganism is maintained in perfectly aerobic conditions whichever of theforegoing methods is employed. It is necessary therefore, after theadditionof the different elements, to maintain the transforming mediumunder conditions of aeration and agitation similar to those used for theinitial culture.

The temperature at which the enzymatic acylating 6 transformation iscarried out is not very critical. Gerierally a temperature between 15and 40 *Crwill be used, theoptimum being in the region of 25 C. l

The transformation of spiramycin I into spiramycins II and III is. arelatively rapid-transformation and a large proportion-of spiramyc'in Iis transformed in 24 hours. In order .to obtain the maximumtransformation yield it is desirable to maintain a sufliciently longcontact between the enzymatic acylating system, the 'spiramycin and theother elements, and several days may then be necessary for the completeevolution of the systemto take place-. After having transformedspiramycin I into spiramycms II and III, the latter can be separated byknown methods such as counter current distribution, chromatography'onaluminua or other adsorbants, or --fra'ct-io'nal crystallisation. Inorder to ascertain the relative proportions ofthe differentspiramycinsduring the course of the transformation or at its end, it isconvenient to apply the method of characterisation by paperchromatographyto the transformingmedium and to compare the chromatogramsobtained with a control chromatogram corresponding to known quantitiescontained in the different spiramycins isolated in the pure state.

The following examples will serve to illustrate the invention.

Example I A 2-litre Erlenmeyerflask is charged with 250 cc. of

the following medium:

Corn-steep (50% dry extract) g 40 Glucose g '20 Sodium chloride g a I 5Magnesium sulphate g 1 Tap water cc 1000 Corn-steep (50% dry extract) g35 Glucose g 50 Sodium chloride g 20 Monopotassium phosphate g 2,Magnesium sulphate g 1 Tapwater cc 1000 The pH is adjusted to 6.8 withThere is then added: Calcium carbonate g 5 Propionamide is then added inthe proportions indicated in the table given below, 'which shows theresults obtained. p v v The 300 cc. Erlenmeyer flasks and their contentsare sterilised for 20 minutes at C. and then, after cool: ing, seededwith 4 cc. of the culture from the 2-litre Erlenmeyer flasks andmaintained at 25 C. on a shaking table. The quantities and the analysisof the antibiotic are carried out on the 6th, 7th and 8th days of theculture in order to determine the maximum activity and respectiveproportions of the three spiramycins.

sodium hydroxide.

. Proportion by weight of Propionamide content of the Maximumsplramyeins (percent) medium, g./l. Activity,

I II III Example If A 170 litre fermentation vessel is charged with:Corn-steep (50% dry extract) kg 4.800 Glucose kg 2.400 Sodium chloride..kg 0.600 Magnesium sulphate kg 0.120 Tap water litres 100 The pH isadjusted to 6.8 by means of sodium hy droxide and the charge iscompleted with:

Calcium carbonate 1 kg 0.600 Soya bean oil litres 0.480

Autolysate of yeast g 300 Glucose g 750 Propionamide g 30 Sodiumchloride g 300 Magnesium sulphate g 15 Monopotassium phosphate g 15 Tapwater 1itres 16.5

The pH is adjusted to 6.5 with sodium hydroxide. The charge is completedwith:

Calcium carbonate g- 75 Soya bean oil cc 60 The medium is sterilised for40 minutes at 120 C. After cooling the volume is 15 litres and the pH is6.8.

The medium is then seeded by the transfer of 2 litres of the inoculumculture from the 170 litre fermentation vessel, then agitated with aturbine turning at 550 rpm. and aerated with l mi /hour of air, andmaintained at 25 C.

From the commencement of the operation the pH falls to reach a value of56 after 60 hours: this first phase corresponds exactly to theconsumption of glucose. The pH then climbs slowly until 90 hours (6.2)and then very rapidly, pasing 7 at 100 hours. The final activity of thebroth is 645 mcg./cm. The respective proportions by weight of the threespiramyeins produced is as follows: I, 12%; II, 13%; III, 75%.

A similar process carried out with the same medium but withoutpropionamide gives after 120 hours an activity of 760 meg/cm. with thefollowing proportions: I, 24%; II, 53%; III, 23%.

Example III The inoculum culture is produced as in Example II. Theproduction culture is carried out this time in an 800 litre fermentationvessel charged with the following medium:

Starch kg The pH is adjusted to 6.7 by the addition of sodium hydroxide(36 B., 1450 cc.), and the charge is completed with: 1 Calcium carbonatekg 2' Soya bean oil cc 1600 The medium is sterilised by bubbling steamthrough it for 40 minutes at 120 C. After cooling, the volume is 400litres and the pH 6.7. The medium is then seeded by a transfer of 40litres of the inoculum culture from the 170 litre fermentation vessel,agitated with a turbine turning at 205 r.p.m., aerated with 15 m. /=hourof air and maintained at 25 C. After 150 hours of culture the activityof the broth is at its maximum: 660 mcg./ 0111. The respectiveproportion by weight of the three spiramycins produced is as follows: I,17%; H, 8%; III, 75%.

Example IV A 2 litre Erlenmeyer flask is charged with 250 cc. of thefollowing medium:

Corn-steep (50% dry extract) g. 40 Starch g 20 Sodium chloride g 5Magnesium sulphate g 1 Monopotassium phosphate g 2 Tap water to make1000 cc.

Corn-steep (50% dry extract) g 45 Glucose g-.. 50

Calcium carbonate ..g 25 Tap water to make 1000 cc. A

The pH of the medium is adjusted to 7 with sodium hydroxide. The mediumis then sterilised for 30 minutes at 120 C. After cooling, the contentof each Erlenmeyer flask is seeded with 4 cc. of the preceding inoculumculture. The culture is then agitated ona shaking table at 25 C. After45 hours the fungal growth is excellent. 20 cc.'of a sterile aqueoussolution containing 12 g.-/l. of pure spiramycin I base and 6 g./l. ofsodium propionate is then added to each Erlenmeyer flask. The agitationof the culture is maintained for 24 hours and then the contents of allthe Erlenmeyer flasks are mixed. The chromatographic analysis of thebroth shows that the latter contains 2.6 g./l. of unconverted spiramycinI and 1.4 g./l. of mixture containing 20% of spiramycin II andspiramycin III.

Example V 300 cc. Erlenmeyer flasks are charged with 40 cc. of thefollowing medium:

Tap water to make 1000 cc.

The pH of the medium is adjusted to 7 before the addition of the calciumcarbonate. The medium is then sterilised for 30 minutes at C. Aftercooling, the contents of each Erlenmeyer flask are seeded with 4 cc. ofan inoculum culture prepared under the conditions of Example IV. Theculture is agitated on a shaking table at 25 C. After 48 hours ofdevelopment, 20 cc. of a sterile aqueous solution containing 12 g./l. ofpure spiramycin I base and 6 g./l. of sodium propionate is added to eachErlenmeyer flask. The culture is allowed to incubate on the shakingtable for 3 days. After this time the contents of the Erlenmeyer flasks.are mixed. The chromatographic analysis of the culture broth shows thatit now contains 2.1 g./i. of unconverted spiramycin I and 1.9 g./1. ofspiramycin I converted into a mixture and cultivated under theconditions described in' Example VIII. After 48 hours of culture theErlenmeyer flasks are divided into two groups. Each Erlenmeyer flask ofthe first group receives 20 cc. of the aqueous solution containing 12g./l. of pure spiramycin I base and 6 g./l. of

containing 6% of spiramycin II and 94% of spiramycin sodium propionate.Each Erlenmeyer flask of the second III. group receives 20 cc. of thesame solution to which has Example VI r I been added 30 g./l. of sodiumchloride. Each group of 300 cc. Erlenmeyer flasks areohargedwith 40 cc,of Ehrlenmeyer g i lsltrated as ii f ranks of the following medium: a aa a 16 c romatograp c ana ysis are as fo ows. Ammomum chlofidg 6Quantity Proportion of spira- Glucose g 25 a. otspiramymycms (percent)Calcium carbonate g 20 m fig Tap water cc 1000 (g./l.) II II TheseErlenmeyer flasks are sterilised, seeded and cul- 15 g 1. 88. 21 79tivated under the conditions described in Example V. 260 23 77 After 48hours of culture 20 cc. of a, sterile aqueous solution containing 9g./l. of pure spiramycin I baseand Example X 11 11 /1. of propionamideis added to each Erlenmeyer Erlenmeyer flasks are charged with 50 of asAfter. three days of incubation the chromatographic folkiwmg medfum'analysis of the brothshows that 1.86 g./l. of spiram-ycin Ammomumchlonde 10 I has been converted into a mixture of 55% of spiramycin I fg 50 IIand.45%v of spirarnycin III. There remains l.14.g./l. 2 Calolumcarbonate 25 of unconverted spiramycin 1. Tap Water to make 1000 vExample VII The Erlemeyer flasks are sterilised, seeded and cultivatedunder the conditions described 1n Example V. A Gilli/wire ofStreptomyce: ambofaclefis N 2420' a After 52 hours of culture eachErlenmeyer flask reout d ditions described 1n Ex p e ceives l cc. of a15% solution of sodium propionate. i Th -R which f base The Erlenmeyerflasks'are then divided into two groups. tams m i 6 of 8021mm prQpwnate'15 Each Erlenmeyer flaskof the first group receives 2 cc. of fioundafter chmmawgrapny of Pl! a 15% solution of pure spiramycin I baseinmethanol. mat there remams of Pncoflvemefiflsplramycm Each Erlenmeyerflask of the second group receives a i and 9 splramycm has f Convertedsolution of the same composition but in acetone. The Into mlxtufeconta'lmng 11% of Splramycm H and 89% culture is allowed toincubate-for72 hours on the shaking of splramycm table. The contents of theErlenmeyer flasks of the same Example VIII group arecombined andanalysed chromatographicallyr 300 cc. Erlenmeyer flasks are chargedwith. 40 CC. of Tne results ofthe transformatlon are the following. thefollowing medium: 40

Quantity Proportion of spira- Ammonium chloride 1O solvent gfgli ig fmycm (Percent) Glucose g 50 fo d Calcium carbonate g. 25 I (EJL) I IIIII Tap water cc 1000 methanol--- 3. 48 24 76 These Erlenmeyer flasksare sterilised, seeded and 0111- a 27 73 tivated under the conditionsdescribed in. Example: V. After 48 hours of culture 20 cc. of a sterileaqueous E a l X1 solution containing 12 g./l. of pure spiramycin I base50 300 cc. Erlenme er flasks are charged as in the precedand 6 of sodiumproplonate is added to; each El ing example, sterilized for 30 minutesat 120 C. and each ineyer flask The Erlenmeyer flasks. are i dmdedseeded with 6 cc. of an inoculum culture prepared as demm 4 groups Thefirst group remain? as It to.the scribed in Example IV. The Erlenmeyerflasks are incuother three groups are added respectwely l m bated on ashaking table at 25 C. and then are divided sulphate, ferrous sulphateand cobalt chloride. The into three groups After 48 hours of cultureeach Erlem addition of these salts is such that each metal occurs in mayflask of first group receives 2 cc of a 15% a concentration of 1millimole per litrein the transforlution in methan 01 of pure,spiramycin I g and 1 cc of mauon medium After three days incubation h a15% aqueous solution of sodium propionate. The same Erlenmeler flasks ofeach group are med and than additions are made to each of the Erlenmeyerflasks of the t i by paper cilmmatography" The two other groups butafter 72 and 96 hours of culture re- Tesum of the analyss are as fOHOWSspectively.. The culture is in each case continued for 3 days after'theaddition'of spiramycin. The contents of ua ti Proportion iSpiratheErlenmeyer flasks of the same group are analysed by 5101mm? Wins(Percent) paper chromatography. The'following table shows the Activatorcm I trans- 1 formed results of the analysts. (g./1.) II III s; a a; renant? 2. 32- 30 Addition of spiramycin, hours cm I trans- 2.80 27 73 70ai H, m

Example IX 3g is g; 300 cc. Erlenmeyer flasks are charged, sterilised,seeded. 28

1 1 Example XII 300 cc. Erlenmeyer flasks are charged with 50 cc. of thefollowing medium:

Ammonium nitrate g 13 Glucose g 50 Calcium carbonate g 25 Tap water tomake 1000 cc.

The Erlenmeyer flasks are sterilised for 30 minutes at 120 C., cooledand seeded with cc. of inoculum culture, the preparation of which isdescribed in Example IV. After three days of culture at 25 C. on ashaking table, 5 cc. of an aqueous solution, containing 100 g./l. ofpure spiramycin I base, propionic acid 38 g./l., cobalt chloridehexahydrate g./l. and the pH of which has been adjusted to 6.5 by theaddition of sodium hydroxide, is added to each Erlenmeyer flask. Theculture is allowed to proceed for 3 further days and the contents of theErlenmeyer flasks are analysed by paper chromatography. 8.7 g./l. ofspiramycin I has been transformed into a mixture containing 9% ofspiramycin II and 91% of spiramycin III.

Example XIII 300 cc. of Erlenmeyer flasks are charged with 50 cc. of thefollowing medium:

The Erlenmeyer flasks are sterilised, seeded and incubated under theconditions described in Example V. After 96 hours of culture, 5 cc. ofan aqueous solution, containing 140 g./l. of a crude spiramycin base(containing respectively 59, 27 and 14% of spiramycins I, II and III),56 g./l. propionic acid, 0.5 g./l. of cobalt chloride hexahydrate and ofwhich the pH was adjusted to 6.5 with sodium hydroxide, is added to eachErlenmeyer flask. The culture is allowed to continue for three days andthe contents of the Erlenmeyer flasks analysed chromatographically. Ofthe 8.26 g./l. of spiramycin I added, 6.3 g./l. had been converted to amixture containing 13% of spiramycin II and 87% of spiramycin III.

Example XIV Two 300 cc. Erlenmeyer flasks are charged with 50 cc. of thefollowing medium:

Ammonium chloride g 10 Glucose g 50 Calcium carbonate g 25 Tap water1000 cc.

The Erlenmeyer flasks are sterilised, seeded and incubated under theconditions described in Example V. After 72 hours of culture thecontents of the Erlenmeyer flasks are centrifuged and the supernatantliquid removed. Half the centrifuged mass is suspended in. 50 cc. of anaqueous solution placed in a 300 cc. Erlenmeyer flask and containing 6g./l. of pure spiramycin I base, 2.3 g./l. propionic acid, 1 g./l. ofcobalt chloride hexahydrate, the pH of the said solution being adjustedto 6.5 by the addi tion of sodium hydroxide. The other half of thecentrifuged mass is suspended in 50 cc. of the aqueous solution of thesame composition but containing in addition 10 g./l. of glucose.

The two Erlenmeyer flasks containing the suspension of mycelium areincubated at 25 C. on a shaking table for two days. After this time thecontents of each of the Erlenmeyer flasks is analysed by chromatography.The results of the transformation are as follows:

A 300 cc. Erlenmeyer flask is charged with 50 cc. of the followingmedium:

Ammonium chloride g 10 Glucose g 50 Calcium carbonate g 25 Tap watercc1000 The Erlenmeyer flask is sterilized, seeded and incubated under theconditions described in Example V. After 72 hours of culture thecontents of the Erlenmeyer flask are centrifuged and the supernatantliquid is eliminated. The centrifuged mass is suspended in 50 cc. of abuffer solution in a 300 cc. Erlenmeyer flask, this buffer solutioncontaining 9.85 g./l. of glacial acetic acid and 4.9 g./l. of sodiumacetate. The pH is 4. It contains in-addition 6.45 g./l. of purespiramycin I hydrochloride and 1 g./l. of cobalt chloride hexahydrate.The Erlenmeyer flask containing the suspension of mycelium is incubatedat 25 C. on a shaking table for hours. The contents of the Erlenmeyerflask are then analysed by paper chromatography. It is thus found that2.40 g./l. of spiramycin I have been converted into a mixture containingof spiramycin II and 10% of spiramycin III.

Example XVI There are placed successively into a 170 litre fermentationvessel:

Tap Water litres The pH is adjusted to 6.7 with 750 cc. of sodiumhydroxide solution (400 g./l. NaOH). There are thenadded:

Calcium carbonate g 600 Soya bean oil cc 60 Silicone anti-foaming agentcc 60 The fermentation vessel and .its contents are sterilised by apassage of steam for 40 minutes at 122 C., which. produces a finalvolume of litres. After cooling and adjusting the temperature to 25 C.,the fermentation vessel is seeded with 250 cc. of an inoculum cultureprepared as has been described in Example IV. The medium is thenagitated by a helix turning at 350 rpm. and aerated with 5 cubic metersof air per hour. After 23 hours of culture the development ofStreptomyces ambofaciens NRRL 2420 is abundant.

A 30 litre fermentation vessel is then charged with the following:

Ammonium chloride g Glucose hydrate g 750 Tap water litres 14 Thefermentation vessel and its contents are sterilised for 40 minutes at122 C. After cooling and adjusting the temperature to 25 C., 375 g. ofcalcium carbonate is added in sterile suspension in 2 litres of waterwhich produces a volume of the medium of 15 litres. The pH is then 7.5.

e rant Spiramycin base g 150 Sodium propionate"; ..g 19 Cobalt chloridehexahydrate g 17 N propionic acid solution cc 300 Distilled Water cc1000 is added to the medium.

The spiramycin base used is a crude product which contains respectively65, 23 and 12% of spiramycins I, II and III.

The culture is then continued under the same conditions of temperature,agitation and aeration for 90 hours. The fermentation vessel is thenemptied and 13.75 litres of broth obtained. Paper chromatography of thebroth showed that all the spiramycin I had been converted into a mixtureof spiramycins II and III, the. proportions respectively being 29 and71% of the final product. Extraction of the broth produces 122 g. ofspiramycin base composed of 25% and 75% of spiramycins II and III,respectively.

We claim:

1. A process for the production of an antibiotic consistingpredominantly of spiramycin III which comprises culturing Streptomycesam bofaciens NRRL 2420 under aerobic conditions on a nutrient culturemedium in the presence ofa propionylating agent selected from the classconsisting of propionic acid, propionic acid salts, propionic esters,propionamide, propanol, valeric acid and valeric acid salts, which isadded to the said medium.

2. A process for the production of an antibiotic consistingpredominantly of spiramycin III which comprises culturing Streptomycesamboyacierzs NRRL 2420 under aerobic conditions on a nutrient culturemedium contain ing a glucidic source of energy, a source of ammoniacalnitrogen and an alkaline substance to hold the pH to values between pHand pH 9' in the presence of a propionylating agent selected from theclass consisting of propionic acid, propionic acid salts, propionicesters, propionamide, propanol, valeric acid and valeric acid salts,which is added to the said medium.

3. A process for the production of an antibiotic con sistingpredominantly of spiramycin III which comprises culturing Streptomycesamboyacz'ens NRRL 2420 under aerobic conditions at 2035 C. on a nutrientculture medium in the presence of a propionylating agent selected fromthe class consisting of propionic acid, propionic acid salts, propionicesters, propionamide, propanol, valeric acid and valeric acid salts,which is added to the said medium.

4. A process for the production of an antibiotic consistingpredominantly of spiramycin III which comprises culturing Streptomycesambofaciens NRRL 2420 under aerobic conditions on a nutrient culturemedium in the presence of a propionylating agent selected from the classconsisting of propionic acid, propionic acid salts, propicnic esters,propionamide, propanol, valeric acid and Valerie acid salts, which isadded to the said medium and in the presence of a metal chloride.

5. A process for the production of an antibiotic consistingpredominantly of spiramycin III which comprises culturing Streptomycesamlbofacz'ens NRRL 2420 under aerobic conditions on a nutrient culturemedium in the presence of a propionylating agent selected from the classconsisting of propionic acid, propionic acid salts, propionic esters,propionamide, propanol, valeric acid v 14 p r and valeric acid salts,which is added to the said. mediuni and in the presence of an activatorselected from-the classc-onsistingof salts of magnesium, iron andcobalt.

6. A process for the production of an antibiotic consistingpredominantly of 'spiramycin III which comprises culturing-Streptomycesambofaciens NRRL 2420 under aerobic conditions on a nutrient mediuminitially free from any spiramycin and from any added acylating agent,using said culture as an inoculum culture to seed a sec-. ond nutrientmedium containing spiramycin'I and a propionylating agent selected fromthe class consisting of propionic acid, propionic acid salts,propionic.esters, propion-amide, propanol, valeric acid and valeric acidsalts and continuing the culture- '7. A process for the production of anantibiotic consisting predominantly of spiramycin III which comprisesculturing Streptomyces umbofaciens NRRL 2420 under aerobic conditions ona nutrient medium initially free from any spiramycin and from any addedacylating agent, using said culture as an inoculum culture to seedasecondnu-trient mediumcontaining a glucidic source of energy', a sourceof ammoniacal nitrogen and an alkaline substance to hold the pH tovalues between pH 5 and pH 9 containing spiramycin I and apropionylating agent selected from the class consisting. of propionicacid,

propionic acid salts, propionic esters, propionamide, propanol, valericacid and valeric acid salts and continuing the culture. I r

8. A process for the production of an antibiotic consistingpredominantly of spiramycin III which comprises culturing Strepto-mycesambofaciens NRRL 2420 under aerobic conditions on a nutrient mediuminitially free from any spiramycinand from any added acylati-n-g agent,

using saidculture .as an inoculum culture to seed a second nutrientmedium containing spiramycin I and a propionylatingagent selected fromthe class consisting of propionic acid, propionic acid salts, propionicesters, pro-. pio-namide, propanol, valeric acid and valeric acid saltsand continuing the culture under aerobic conditions at 20-35 C. Y 7 p.9. A process for the production of an antibiotic con.- sistingpredominantly of spiramycin III which comprises culturingStreptomycesambofaciens NRRL 2420 under aerobic conditions on a nutrient mediuminitially free .from any spiramycin and fromany added acylating agent,using said culture as an inoculum culture to seed a second nutrientmedium containing spiramycin I and a propionylating agent selected fromthe class consisting of propionic acid, propionic acid salts, propionicesters, propionamide, propanol, valeric acid and valeric acid salts andfurther containing a metal chloride and continuing the culture.

10. A process for the production of an antibiotic consistingpredominantly of spiramycin III which comprises culturing Streptomycesambofaciens NRRL 2420 under aerobic conditions on a nutrient mediuminitially free from any spiramycin and from any added acylating agent,

using said culture as an inoculum culture .to seed a second nutrientmedium containing spir-amycin I and a propionylating agent selected fromthe class consisting of propionic acid, propionic acid salts, propionicesters, propionamide, propanol, valeric acid and valeric acid salts andfurther containing an activator selected :fiom .the class consisting ofsalts of magnesium, iron and cobalt and continuing the culture.

11. A process for the production of an antibiotic consistingpredominantly of spiramycin III which comprises culturing Streptomycesambofaciens NRRL 2420 under aerobic conditions on a nutrient mediuminitially free from any spiramycin and from any added acylating agent,separating the mycelium from said culture, using said mycelium as aninoculum culture to seed a second nutrient medium containing spiramycinI and a propionylating agent selected from the class consisting ofpropionic :acid, propionic acid salts, propionic esters, pro- 15pionamide, propanol, valeric acid and valeric acid salts and continuingthe culture.

12. A process for the production of an antibiotic consistingpredominantly of spiramycin III which comprises culturing Streptomycesambofaciens NRRL 2420 under aerobic conditions on a nutrient mediuminitially free from any spiramycin and from any added acylating agent,separating the mycelium from said culture, using said mycelium as aninoculum culture .to seed a second nutrient medium containing a glucidicsource of energy, a source of ammoniacal nitrogen and an alkalinesubstance to hold the pH to values between pH and pH 9 and containingspiramycin I and a propionylating agent selected from the classconsisting of propionic acid, propionic acid salts, propionic esters,propionamide, propanol, valeric acid and Valerie acid salts andcontinuing the culture.

13. A process for the production of an antibiotic consistingpredominantly of spiramycin III which comprises culturing Streptomycesambofaciens NRRL 2420 under aerobic conditions on a nutrient mediuminitially free from any spiramycin and from any added acylating agent,separating the mycelium from said culture, using said mycelium as aninoculum culture to seed a second nutrient medium containing spiramycinI and a propionylating agent selected from the class consisting ofpropionic acid, propionic acid salts, propionic esters, propionamide,propanol, valeric acid and valeric acid salts and continuing the cultureunder aerobic conditions at 20-35 C.

14. A process for the production of an antibiotic consistingpredominantly of spiramycin III which comprises culturing Streptomycesambofaciens NRRL 2420 under aerobic conditions on a nutrient mediuminitially free from any spiramycin and from any added acylating agent,separating the mycelium from said culture, using said mycelium as aninoculum culture to seed a second nutrient medium containing spiramycinI and a propionylating agent selected from the class consisting ofpropionic acid, propionic acid salts, propionic esters, propionamide,propanol, valeric acid and valeric acid'salts and further containing ametal chloride and continuing the culture.

15. A process for the production of an antibiotic consistingpredominantly of spiramycin III which comprises culturing Streptomycesambofaciens NRRL 2420 under aerobic conditions on a nutrient mediuminitially tree from any spiramycin and from any added acylating agent,separating the mycelium from said culture, using said mycelium as aninoculum culture to seed a. second nutrient medium containing spiramycinI and a propionylating agent selected from the class consisting ofpropionic acid, propionic acid salts, propionic esters, propionamide,propanol, valeric acid and valeric acid salts and further containing anactivator selected from the class consisting of salts of magnesium, ironand cobalt and continuing the culture.

16. A process for the production of an antibiotic consistingpredominantly of spiramycin III which comprises culturing Streptomycesambofaciens NRRL 2420 under aerobic conditions at 20-35 C. on a nutrientmedium initially free from any spiramycin and from any acylating agent,using the said culture as an inoculum culture to seed a second nutrientmedium which includes a glucidic source of energy, a source ofammoniacal nitrogen, an alkaline substance to hold the pH to valuesbetween pH 6.5 and 7.5, a metal chloride, an activator selected from theclass consisting of salts of magnesium, iron and cobalt, spiramycin Iand a propionylating agent selected from the class consisting ofpropionic acid, propionic acid,

salts, propionic esters, propionamide, propanol, valeric acid andvaleric acid salts and continuing the culture under aerobic conditionsat 20-35 C.

References Cited in the file of this patent UNITED STATES PATENTS OTHERREFERENCES Pinnert: Annals, Inst. Pasteur, vol. 87, 1954, page 702.

Corbaz et al.: Stefiiwechselprodulcte von Actinomyceten, HelveticaChemica Acta, vol. 39, No. 32, February 1, 1956, pages 304-317.

1. A PROCESS FOR THE PRODUCTION OF AN ANTIBIOTIC CONSISTINGPREDOMINANTLY OF SPIRAMYCIN III WHICH COMPRISES CULTURING STREPTOMYCESAMBOFACIENS NRRL 2420 UNDER AEROBIC CONDITIONS ON A NUTRIENT CULTUREMEDIUM IN THE PRESENCE OF A PROPIONYLATING AGENT SELECTED FROM THE CLASSCONSISTING OF PROPIONIC ACID, PROPIONIC ACID SALTS, PROPIONIC ESTERS,PROPIONAMIDE, PROPANOL, VALERIC ACID AND VALERIC ACID SALTS, WHICH ISADDED TO THE SAID MEDIUM.